Optical system for multiple reactions

ABSTRACT

Optical systems, and corresponding methods, for multiple reactions are provided. The optical systems are in a fixed position relative to a thermal assembly and include at least one array of excitation sources (e.g., light emitting diodes (LEDs)) configured to output excitation energy along an excitation optical path. In addition, a detector configured to receive emission energy along a detection optical path in the same plane as the excitation optical path is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/841,725 filed on Jul. 22, 2010, allowed, which claims priority toU.S. Provisional Application No. 61/240,951 filed on Sep. 9, 2009 andU.S. Provisional Application No. 61/296,847 filed on Jan. 20, 2010, eachof which is hereby incorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

This application relates to thermocylers. More specifically, thisapplication relates to systems and methods for detecting amplificationof molecules using the polymerase chain reaction and other reactions.

2. Description of the Related Art

The advent of Polymerase Chain Reaction (PCR) in 1983 has revolutionizedmolecular biology through vastly extending the capability to identify,manipulate, and reproduce genetic materials such as DNA. Now, PCR isroutinely practiced in medical and biological research laboratories fora variety of tasks, such as the detection of hereditary diseases, theidentification of genetic fingerprints, the diagnosis of infectiousdiseases, the cloning of genes, paternity testing, and DNA computing.The method has been automated through the use of thermal stable DNApolymerases and machines capable of heating and cooling genetic samplesrapidly, commonly known as thermal cyclers.

The optical measurements useful for interrogating these reactions caninvolve the measurement of fluorescence. To measure fluorescence,excitation light is directed at the samples in the sample vessels, andlight emitted from the fluorophores in the samples is detected. It isoften desirable that the transfer of light from the light source to thewells be carried out effectively and efficiently. Optical systems fordirecting light to sample plates is known, for example, as described inU.S. Pat. Nos. 6,942,837, 7,369,227, 6,852,986, and 7,410,793. Whileoptical systems for directing light to sample vessels in plates anddetecting light from the sample vessels have been developed in the art,there remains a need for optical systems which can do so moreeffectively and efficiently.

SUMMARY

The systems and methods described in the claims each have severalfeatures, no single one of which is solely responsible for its desirableattributes. Without limiting the scope of this invention, certainfeatures will now be briefly discussed.

One aspect of the disclosure is a system that includes a thermalassembly configured to hold a sample plate. The system also includes anoptical assembly in a fixed position relative to the thermal assembly.The optical assembly includes at least one array of excitation sourcesconfigured to emit excitation energy along an excitation optical path,and a detector configured to receive emission energy along a detectionoptical path. The excitation optical path and the detection optical pathare in the same plane.

In certain implementations, the system further includes one or more amultifunction mirror; an excitation source assembly comprising an arrayof excitation sources; an emission filter slide; a detection lensassembly; a set of excitation optics; or a control assembly. In some ofthese implementations, the system includes a multifunction mirror, twoexcitation source assemblies, two sets of excitation optics and adetector. According to various implementations, in the excitationoptical path, an excitation source assembly and a set of excitationoptics is positioned on one side of the multifunction mirror and anotherexcitation source assembly and another set of excitation optics ispositioned on the other side of the multifunction mirror. Additionally,in a number of implementations, the excitation source assembly ispositioned in the excitation optical path and wherein the excitationsource assembly includes individual light emitting diodes (LEDs)corresponding to individual wells on the sample plate. In someimplementations, the excitation source assembly further includes an LEDarray comprising the LEDs. In accordance with certain implementations,the excitation source assembly further includes a lenslet arrayincluding individual lenslets corresponding to individual LEDs in theLED array.

In certain implementations, the multifunction mirror is positioned inthe excitation optical path to direct excitation energy from individualLEDs to the corresponding wells of the sample plate and is furtherpositioned in the detection optical path to direct emission energy fromthe sample plate to a detector. In a number of implementations, thedetector is positioned in the detection optical path and is capable ofdetecting emission energy from the sample plate. In variousimplementations, the emission energy is fluorescent emission and thefluorescent emission is directed to the detector from the multifunctionmirror. In some implementations, the system further includes an emissionfilter slide including at least one emission filter. According tocertain implementations, the emission filter slide is positioned in thedetection optical path between the multifunction mirror and thedetector. In accordance with a number of implementations, the emissionfilter slide includes four emission filters, wherein each emissionfilter filters a different wavelength. In various implementations, thesystem further includes an emission filter motor for moving the emissionfilter slide.

In certain implementations, the set of excitation optics includes anexcitation filter and two lenses. In some implementations, the systemfurther includes a movable lid that is positioned to move around theoptical assembly and wherein the movable lid includes a heated lidconfigured to mate with the sample plate. In some implementations, thethermal assembly includes a thermal block that includes the sampleplate. According to a number of implementations, a liquid compositionoccurs within the thermal block and external to the sample plate.

In certain implementations, the system further includes: (a) twoexcitation source assemblies positioned in the excitation optical path,wherein an excitation source assembly includes individual light emittingdiodes (LEDs) corresponding to individual wells on the sample plate; (b)a multifunction mirror positioned in the excitation optical path todirect excitation energy from individual LEDs to the correspondingindividual wells of the sample plate and wherein the multifunctionmirror is further positioned in the detection optical path to directfluorescence emission from the sample plate to a detector; and (c) anemission filter slide positioned in the detection optical path betweenthe multifunction mirror and the detector, wherein the emission filterslide includes at least one emission filter. In accordance with a numberof implementations, the excitation optical path comprises two excitationsource assemblies and two sets of excitation optics. In some of theseimplementations, each set of excitation optics comprises an excitationfilter and two lenses.

In some implementations, each excitation source assembly includes an LEDbackplate to which an LED array including the LEDs is mounted, a lensletarray including individual lenslets corresponding to individual LEDs inthe LED array, wherein the lenslet array is positioned to transmit theexcitation energy from the LEDs on the LED array to the multifunctionmirror and an excitation source cooling portal positioned to cool theLED backplate. In some implementations, the emission filter slideincludes four emission filters, each emission filter filtering adifferent wavelength. In accordance with various implementations, thesystem further comprising an emission filter motor for moving theemission filter slide. According to a number of implementations, thesystem further includes at least one detection lens assembly positionedbetween the emission filter slide and the detector. In someimplementations, the system further includes a movable lid that ispositioned to move around the optical assembly and wherein the movablelid includes a heated lid configured to mate with the sample plate.

In certain implementations, the thermal assembly includes a thermalblock comprising the sample plate. In some implementations, a liquidcomposition occurs within the thermal block and external to the sampleplate.

In another aspect, a system comprises an optical assembly in a fixedposition relative to a thermal assembly configured to hold a sampleplate, wherein the system comprises a movable lid that moves around theoptical assembly, and wherein the movable lid comprises a heated lidconfigured mate with the sample plate. In some instances, the opticalassembly comprises at least one excitation optical path and at least ondetection optical path, wherein the excitation optical path and thedetection optical path are in the same plane.

In some instances, the optical assembly comprises a multifunction mirrorthat directs excitation energy to the sample plate from the at least oneexcitation optical path and that directs emission energy from the sampleplate to the detection optical path. The optical assembly can compriseat least one excitation source, wherein the excitation source comprisesone LED corresponding to each well on the sample plate. In someinstances, the optical assembly comprises a detector capable ofdetecting fluorescent emission from the sample plate.

In some instances, the thermal assembly comprises a closed liquidreservoir and a stirrer in thermal contact with a heater. In someinstances, the sample plate comprises 48 wells.

In some instances, the movable lid is a hinged lid. The movable lid canfurther comprise an optical component, for example, a Fresnel lens. Insome instances, the movable lid further comprises a spring-like orflexible component configured to improve the thermal contact of theheated lid to the sample plate.

In yet another aspect, a system comprises an optical assembly configuredto excite a biological assay and to detect emission from the biologicalassay, wherein the optical assembly comprises at least one excitationoptical path and at least on detection optical path, wherein theexcitation optical path and the detection optical path are in the sameplane. In some instances, the optical assembly comprises at least oneexcitation source, wherein the excitation source comprises one LEDcorresponding to each well on the sample plate. In some instances, theoptical assembly comprises a detector capable of detecting fluorescentemission from the sample plate.

In some instances, the optical assembly comprises a multifunction mirrorthat directs excitation energy to the sample plate from the at least oneexcitation optical path and that directs emission energy from the sampleplate to the detection optical path.

The system can further comprise a thermal assembly configured tothermally cycle the biological assay. In some instances, the thermalassembly comprises a closed liquid reservoir and a stirrer in thermalcontact with a heater.

For purposes of summarizing the disclosure, certain aspects, advantagesand novel features of the inventions have been described herein. It isto be understood that not necessarily all such advantages may beachieved in accordance with any particular embodiment of the invention.Thus, the invention may be embodied or carried out in a manner thatachieves or optimizes one advantage or group of advantages as taughtherein without necessarily achieving other advantages as may be taughtor suggested herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Many features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the invention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which manyprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 illustrates an exemplary excitation optical path of the opticalsystem as described herein.

FIG. 2 illustrates an exemplary embodiment of the detection optical pathof the optical system as described herein.

FIG. 3 shows another view of the detection optical path from the planeof the multifunction mirror.

FIG. 4 displays an exemplary optical assembly as described hereincomprising both detection optics and excitation optics in the sameplane.

FIG. 5A is a side view of an exemplary optical assembly as describedherein.

FIG. 5B is a side view of an exemplary optical assembly as describedherein.

FIGS. 6 and 7 display a view of an exemplary excitation source assemblyherein.

FIGS. 8 and 9 display another exemplary optical assembly as describedherein comprising both detection optics and excitation optics in thesame plane.

FIG. 10 provides a zoomed view of the multifunction mirror and theexcitation and detection optics.

FIG. 11 illustrates a sample image from a 48-well sample plate asexcited and detected using an optical system as described herein

FIG. 12 illustrates an exemplary optical assembly as described hereincomprising a multifunction mirror.

FIG. 13 shows a top view of the optical system over the sample plate.

FIG. 14 demonstrates the detection optical path of the optical assembly.

FIG. 15 illustrates an excitation source assembly as described herein.For example, the assembly can comprise an LED array.

FIG. 16 illustrates an exemplary optical assembly as described hereincomprising a multifunction mirror, excitation optics, and an excitationsource assembly.

FIG. 17 illustrates an excitation source board on which an array of LEDscan be mounted.

FIG. 18 illustrates an exemplary LED as can be incorporated into asystem of the invention.

FIG. 19 shows an exemplary thermal cycling device with optical system asdescribed herein.

FIG. 20 demonstrates an exemplary thermal cycling device with opticalsystem as described herein with the hinged lid open.

FIG. 21 is a planar view of an exemplary device as described herein.

FIG. 22 illustrates an exemplary device as described herein comprising adevice body and not displaying the cover to the hinged lid.

FIG. 23 illustrates a hinged lid plate that can be coupled with a hingedlid that covers the optical assembly.

FIG. 24 demonstrates a thermal block and a compression plate of a systemas described herein.

FIG. 25 displays a system herein comprising an optical assembly and ahinged lid plate.

DETAILED DESCRIPTION

Described herein are a devices, methods, and systems for measuringmultiple chemical reactions in multiwell plates. An optical assembly ofsystems herein can comprise multiple light sources, such as lightemitting diodes (LEDs). In the optical assembly, each light source, forexample an LED, corresponds to a sample vessel on the multiwell plate.

Example embodiments of the invention described herein utilize particulararrangements of optical elements such as lenses, filters, andreflectors. There are many other optical arrangements which can beimplemented in order to carry out or construct aspects of the disclosurethat would be understood by one of ordinary skill in the art fordirecting the light beams to the corresponding sample vessels and fordirecting light from the sample vessels to a detector.

Where the optical system has an excitation source array, a detector, andthe appropriate filters, lenses, and reflectors, the system can be usedas a fluorometer. A fluorometer provides excitation light to a sampleand detects the light emitted from fluorescent entities within thesample.

Also disclosed herein are systems for the controlled heating of samplessuch as biological samples for thermal cycling reactions. The devicesherein can offer improved temperature uniformity and distribution tocurrent technology in the art. Temperature uniformity can be highlydesirable in PCR reactions, for example, where a plurality of samples ina plurality of reaction containers are advantageously cooled or heatedsimultaneously.

In addition to heating of PCR samples, the devices and methods hereincan be used widely in the field of biotechnology and chemistry. Examplesinclude but are not limited to incubations of enzymatic reactions suchas restriction enzymes, biochemical assays and polymerase reactions;cell culturing and transformation; melting of nucleic acids;hybridization; and any treatment requiring precise temperature control.Based on the present disclosure, one of ordinary skill in the art canreadily adapt the disclosed technology to various analyses of biologicall chemical samples which require accurate temperature control.

In some instances, a system as described herein can further comprise anoptical assembly having a light source and an optical detector, whereinthe optical assembly is positioned such that light from the light sourceis directed into the sample holder, and light from the sample holder isdetected by the detector. The detector of the optical assembly cancomprise a PIN photodiode, a CCD imager, a CMOS imager, a line scanner,a photodiode, a phototransistor, a photomultiplier or an avalanchephotodiode. The excitation source can comprises one or more LEDs, laserdiodes, vertical cavity surface emitting lasers (VCSELs), verticalexternal cavity surface emitting lasers (VECSELs), or diode pumped solidstate (DPSS) lasers. In some embodiments, the optical elements can bearrays of light emitting diodes (LEDs). LEDs have advantages as lightsources for the optical systems provided herein in that they are small,relatively inexpensive, generate relatively low heat, and can providelight in the spectral ranges required for measuring samples, for exampleby fluorescence.

An optical detector as described herein can comprise a plurality ofoptical detectors, wherein at least one optical detector corresponds toa sample well in a sample microplate.

An apparatus herein can also further comprise a control assembly whichcontrols the apparatus, the light source, and the detector. In someinstances, the control assembly comprises a programmable computerprogrammed to automatically process samples, run multiple temperaturecycles, obtain measurements, digitize measurements into data and convertdata into charts or graphs.

FIG. 1 illustrates an example excitation optical path of the opticalsystem as described herein. As shown in FIG. 1, the excitation path cancomprise two LED arrays 102 mounted on a backplate 104. As describedherein, the LED array assembly can be cooled by cooling the backplate104 on which the LED arrays 102 are mounted. In an embodiment, the LEDarrays 102 emit the same color or wavelength of excitation energy. Inanother embodiment, the LED array 102 emits a different color orwavelength of excitation energy, for example, one array emits blueexcitation energy and the other array emits green excitation energy. Theexcitation energy from the LED array 102 travels through a lens array106. In an embodiment, the lens array 106 is a lenslet array, comprisinga lenslet corresponding to each LED in the LED array 102, for example,48 lenslets for 48 LEDs. After travelling through the lens array, theexcitation optical path travels through excitation optics 108 which caninclude without limitation filters, lens, or fiber optics. Theexcitation energy 110 is directed by the multifunction mirror 112towards the thermal block. As demonstrated in FIG. 1, the multifunctionmirror 112 can have at least two faces in the excitation path, each facecorresponding to an LED array. A Fresnel lens 114 or other opticaldevice can be mounted above or coupled to a heated lid cover 116 for thesample plate 118. In an embodiment, the Fresnel lens 114 and the heatedlid are incorporated into a hinged lid that moves over and around astationary optical assembly. The excitation energy 110 is directed intothe sample plate 118 which is mounted into a thermal block as shown inFIG. 1. In an embodiment, the optical assembly comprises two fixed LEDsystems and four emission filters to support standard dyes, includingwithout limitation SYBR Green I, FAM, HEX, ROX, and Cy5.

FIG. 2 illustrates an example embodiment of the detection optical pathof the optical system as described herein. As demonstrated by FIG. 2,emission energy 201 is emitted from the sample in the sample wells ofthe sample plate 204, for example, by fluorescence. The emission energy201 travels back through the heated lid cover 202 of the sample plate204, through the Fresnel lens 206 and to the multifunction mirror 208.In an embodiment as demonstrated herein, the multifunction mirror 208 isthe same multifunction mirror 112 as demonstrated in FIG. 1, wherein themultifunction mirror is a three-sided mirror to allow both theexcitation optical path and detection optical path to be in the sameplane in the optical assembly. In another embodiment, the multifunctionmirror 208 is a different mirror than that in the excitation opticalpath. As demonstrated in FIG. 2, the detection optical path travelsthrough the detecting optics 210 which can include without limitationlenses, fiber optics, and optical filters. The optical path can thenoptionally travel through an optical filter. In the example of FIG. 2,the optical detection filter 212 is a single longitudinal device withmultiple filters that can be moved in the path to filter differentwavelengths of energy. For example, depending on the wavelength of thedetection dye used in the sample plate, the optical detection filter 212can be changed to filter out any excess noise not in the color range ofthe dye. The detection optical path ends at the detector 214, where theemission energy from the sample plate can be detected to complete anassay with the system as described herein. The detector 214 ispositioned in the detection optical path and is capable of detectingemission energy from the sample plate. In some instances, the emissionenergy is fluorescent emission and the fluorescent emission is directedto the detector from the multifunction mirror 208.

FIG. 3 demonstrates another view of the detection optical path from theplane of the multifunction mirror 208. The excitation assembly,including an excitation source assembly 304 and excitation optics 306,is in the same plane as the detection optics 210 in FIG. 3. Asillustrated, the excitation assembly is positioned in the excitationoptical path. The excitation assembly provides excitation energy throughthe excitation optics 306 to the multifunction mirror 208 which directsthe energy to the sample plate. In some implementations, the componentsof the excitation assembly are substantially similar to theircorresponding components described above with reference to FIG. 1.Emission energy returns from the samples in the sample plate through themultifunction mirror 208, which then sends the emission energy through aseries of detection optics 210 in the detection optical path including adetection filter of the detection filters 212, as described above, tothe detector 214. Thus, the multifunction mirror directs excitationenergy to the sample plate from the excitation optical path and directsemission energy from the sample plate to the detection optical path. Inaddition, as illustrated, the multifunction mirror is positioned in theexcitation optical path to direct excitation energy from each LED to thecorresponding well of the sample plate and is further positioned in thedetection optical path to direct emission energy from the sample plateto the detector.

FIG. 4 displays an example optical assembly 402 as described hereincomprising both detection optics 404 and excitation optics 406 in thesame plane. In particular, a fixed excitation source assembly 408directs light through the excitation optics 406 in the same plane aslight is received by the fixed detection optics 404. As illustrated inFIG. 4, two sets of excitation source assemblies 408 can generateoptical signals in the same plane as one or more optical signalsreceived by the detector 410. Moreover, the source assemblies 408 andthe detection optics 404 and/or the detector 410 are also in the sameplane in the illustrated embodiment. As displayed, the excitation sourceassembly 408 comprises an LED array. The LED array is mounted on an LEDbackplate 412. In some instances the LED backplate 412 comprisesaluminum. In some instances, the LED backplate 412 is heat conductiveand transfer heat away from the LED array. Also demonstrated in FIG. 4are excitation optics 406, detection optics 404 including emissionfilters, and a detector 410. In some instances, the emission filters aremounted on an emission filter slide 414. As illustrated, the emissionfilter slide 414 is positioned in the detection optical path between themultifunction mirror and the detector 410. The emission filter slide 414can be moved by an emission filter motor 416, for example as shown inFIG. 4. In some instances, the emission filter slide 414 comprises 5emission filters for filtering 5 different wavelengths. In someinstances, the emission filter slide 414 comprises 1, 2, 3, or 4emission filters. In some instances, the emission filter slide 414comprises 6 or more emission filters. In some instances, the emissionfilter slide 414 comprises emission filters, wherein each emissionfilter filters a different wavelength. In some instances, the emissionfilter slide 414 comprises at least 2 emission filters that filter thesame wavelength. As shown in FIG. 4, the detector 410 is mounted afterthe detection optics 404.

FIGS. 5A-B illustrate a side view of an example optical assembly 502 asdescribed herein. FIG. 5A displays an optical assembly comprising amultifunction mirror 504 in the excitation optical path. Themultifunction mirror 504 directs light from the excitation source (forexample, LED array) in an excitation source assembly 506 to a sampleholder and also direct light from the sample holder to the detector. Theexcitation optical path from the excitation source to the multifunctionmirror 504 comprises excitation optics 507 that are capable of directingthe light, filtering the light, or shaping the light. In some instancessuch as in FIG. 5A, the excitation source assembly 506 comprises an LEDarray 508 mounted on an LED backplate 510 and a lenslet array 512 fordirecting the energy from the LED array 508 to the excitation optics 507in the optical path. FIG. 5B illustrates another example side view of anoptical assembly herein, displaying the detection optical path. Thedetection optical path comprises an emission filter slide 520 comprisingemission filters for different detection wavelengths and detectionoptics 522 that are capable of directing the light, filtering the light,or shaping the light to the detector 524. The emission filter slide 520can be moved by an emission filter motor as demonstrated in FIG. 5B.FIG. 5B also displays a back view of the excitation source assembly. Insome instances, the excitation source assembly comprises an LED arraymounted on an LED baseplate 528. The excitation source assembly cancomprise an excitation cooling portal 530, which allows for cooling ofthe excitation source. When the excitation is an LED array mounted on abaseplate 528, the LED array can be actively cooled through theexcitation cooling portal 530.

FIGS. 6 and 7 display a view of an example excitation source assemblyherein. The excitation source assembly 602 comprises an LED array 604,an LED baseplate 606, a lenslet array 608, and an excitation sourcecooling portal 610. In some instances, the excitation source assembly602 also comprises an adjustment assembly 612 that permits moving theassembly in three dimensions in order to position the excitation sourceassembly 602. The adjustment assembly 612 can be moved to align theexcitation optical path with the excitation optics of the opticalassembly 602.

FIGS. 8 and 9 display another example optical assembly 802 as describedherein comprising both detection optics 804 and excitation optics 806 inthe same plane. In some instances, the excitation optics 804 comprise anexcitation filter 808 and two lenses 810. In certain instances, theexcitation filter 808 is positioned between the two lenses 810. Theexcitation filter 808 is chosen based on the wavelength of theexcitation light source and the assay to be run in the sample holder. Inthe examples of FIGS. 8 and 9, the optical assembly 802 comprises twoexcitation source assemblies 812 and two sets of excitation optics 806.In some instances, the excitation source assemblies 812 provide twodifferent wavelengths of excitation light and the excitation optics 806can correspond to the different wavelengths. In some instances, theexcitation source assemblies 812 provide the same wavelengths ofexcitation light. Also demonstrated in FIG. 9 are detection optics 804comprising two detection lens assemblies 814 and an emission filterslide 816 comprising emission filters 828. The emission filter slide 816can be moved by an emission filter motor 818. This can change whichemission filter 828 is in the detection path. A detector 820 and amultifunction mirror 822 are also illustrated in FIGS. 8 and 9.

FIG. 10 provides a magnified view of the multifunction mirror 822 andthe excitation and detection optics 806, 804, respectively, illustratedin FIGS. 8 and 9. The multifunction mirror 822 allows for the detectionand excitation optics 806, 804, respectively, to be in the same plane asthe excitation source assembly 812 and/or the detector 820 of theoptical assembly 802. The excitation optics 806 comprise an excitationfilter 808 and two lenses 810. The detection optics in FIG. 10 comprisea lens assembly 814. As illustrated, the system includes themultifunction mirror 822, the two excitation source assemblies 812, andthe two sets of excitation optics 806; in the excitation optical path,one excitation source assembly and one set of excitation optics ispositioned on one side of the multifunction mirror 822 and anotherexcitation source assembly and another set of excitation optics ispositioned on another side of the multifunction mirror 822.

In real-time PCR, fluorescence measurements of a sample mix are madeafter the PCR amplification step (or sometimes more often) in order totrack the amount of PCR product generated. Details vary according to thePCR chemistry being used, but typically a baseline fluorescence levelincreases to a final fluorescence level which is several orders ofmagnitude more intense at the end of the run, the increase beingexponential in the interesting portion of the run. Quantitationtypically consists of determining the fractional cycle number at whichthe fluorescence of the reporter dye increases by a predetermined smallfraction of the baseline level; this requirement puts a premium on thestability of the fluorescence signal over many cycles, as well asrequiring sufficiently low random noise. Noise sources intrinsic to thePCR process, such as starting copy number statistics and amplificationnoise, place a lower limit on the degree to which improved radiometricand other detection system noise can improve overall quantitativeprecision.

In the real-time PCR systems described herein, many (for example, 48)PCR samples are amplified simultaneously in a 2-D plate format, and thedetection system accommodates this parallelism. In some embodiments,ease of alignment and calibration, measurement speed, robustness andreliability are important drivers in addition to adequate quantitativeperformance. In many cases, multiple dyes (for example, up to about 4)are measured in each sample, either to track multiple reactions withineach well (multiplexing) or to accommodate various multi-dyechemistries. This requirement significantly complicates theoptomechanical design. A number of commercial real-time PCR instrumentshave been marketed for many years, using a variety of detection schemes.

In some instances, a system herein has minimal moving parts and theoptical system is fixed relative to the thermal block. In addition, adevice, as demonstrated herein can be configured to be robust in regardsto shock and vibration during shipment or in operation. In someinstances, a system herein for real-time PCR can operate at a quickspeed, and have very good reproducibility and stability. In someembodiments, switching from dye channel to dye channel can be doneinstantaneously, allowing measurements of different dyes to beinterleaved when simultaneity is important.

Excitation and emission beams at the sample in fluorescence detectionsystems can overlap using beam splitters. These beam splitters may beeither dichroic (spectrally selective) or neutral. Beam splitters offerthe advantage of maximizing the excitation and emission etendues whereetendues are limited by the well geometry rather than source or detectorfactors. Also, dichroic splitters allow nearly full transmission forboth excitation and emission beams when designed for a wavelengthtransition between the dye excitation and emission bands, and cantherefore be radiometrically efficient. However, dichroic beamsplittershave a couple of problems, such as: (a) typically operating at 45degrees, they exhibit very high angular sensitivity of the transitionwavelength, making them hard to design for a large field (many well)system, and (b) usually only a single excitation wavelength can beaccommodated with a given dichroic. The latter restriction means thatfor a multiple dye system, the excitation should be at a singlewavelength, to the blue of the shortest wavelength dye, or else thebeamsplitter should be changed (via mechanical motion) for each dyemeasurement, or finally a complex set of sequential dichroicbeamsplitters should be engineered. Using a single excitation wavelengthmeans that some of the dyes are very inefficiently excited, and that anunfavorable ratio of emission intensities is likely—making dye-dyecrosstalk effects more difficult to untangle. Neutral beamsplitters(such as 50%/50%) do not suffer these disadvantages, but do result in atleast 4× smaller signal levels. All beamsplitter designs also place anadditional optical component, generally with two surfaces, in the commonbeam paths. This component is a potential source of parasiticfluorescence and of direct scattering from the excitation source intothe detection channel.

An alternative approach which avoids most of these problems is toseparate the excitation and emission beams geometrically, and perhapsalso the excitation and/or emission beams corresponding to different dyechannels. Since the beams should overlap at the sample, the separationis normally done at the complementary stop, also referred to as themultifunction mirror herein. In other words, the solid angle space asseen from the sample volume is divided up geometrically among n=2 ormore beams. This results in a signal reduction of ˜n—assuming that thesource and detection optics, including any filters, had sufficientetendue to match the full etendue of the sample well in the first place.If not, the signal reduction factor is lower than n. This radiometrichit is similar to that using neutral beamsplitters, but with the verylarge advantage that the etendue requirements for the source anddetection optics (including their lenses and filters) are much smaller.This means cheaper, smaller optics, and often less difficulty withaberration control. The beamsplitters themselves are also eliminated, ofcourse, although generally some pupil division optic (mirror or prism)would still be implemented.

In many cases, plate readers employing simultaneous imaging of the fullplate use a single relatively powerful source to illuminate all wells.This can be a laser, filtered arc or tungsten lamp, or a high power LED,among other possibilities. From the standpoints of cost, longevity, heatand power consumption, stability, and timing control; LED sources arehighly desirable. While it is possible to employ multi-die high poweredLED sources capable of illuminating a full well plate with somewhatadequate irradiance, an attractive and obvious alternative is to use anarray of low cost, low power LEDs whose dies can be imaged directly intothe sample wells.

In some instances, by exercising independent control of multipleexcitation sources, large system advantages can be obtained, asdescribed below. The basic advantages of array illumination are that lowcost devices, potentially available in a wider variety of wavelengths tomatch the excitation spectra of more dyes, can be used, and that byconfining illumination to the active regions of the individual wells,one can avoid many problems of well-well overlap and crosstalk,including effects of parasitic fluorescence in construction materials.The overall power efficiency can also be improved; less total inputpower per unit of useful illumination.

Issues with multiwell real-time PCR include a limited dynamic range.Different wells may have fluorescent emission fluxes which differ by upto 2 to 3 orders of magnitude, and similar ratios may apply betweenindividual dyes in the same well. This means that stray light from wellA to well B should either be very tightly controlled or somehowcompensated for in data analysis, lest large error appear in PCRresults. Only a small amount of crosstalk, can have very serious effectson baseline wells in comparison to typical radiometric noise levelswhich otherwise determine quantitative precision. Stray light can be dueto scattering within the well plate plastic itself, or from opticalelements in the light path above the well plate. Multiple specularreflections from optical surfaces can also produce significantcrosstalk, often between highly-separated wells.

A second effect of the dynamic range encountered across a plate is thatin order to achieve adequate signal to noise ratios at the variousfluorescence intensities, CCD or CMOS array detectors are generallyoperated at several integration times, a relatively long one for weakerwells, together with at least 1 or 2 shorter exposures to accommodateintense wells without detector pixel saturation. This results inadditional data transfers and additional measurement time. Moresignificantly, the intense wells will strongly saturate the detectorduring the longest exposure times, meaning that array detectors havingextremely efficient anti-blooming performance may be chosen. Also, ofcourse, the purely optical part of the cross-talk cannot be eliminatedin this way in any case. It is possible (and may be necessary in someinstances) to provide software compensation for crosstalk, based onmeasured system crosstalk data.

Independent time gating of excitation LEDs allows a single arraydetector integration time can be used, with the temporal duty factor ofthe LEDs adjusted per-well to achieve nominally similar exposures foreach well (say 50-75% of full well for pixels in the middle of each wellimage). Thus, wells emitting only baseline fluorescence levels can havetheir LEDs turned on 100% of the time (one die at a time), whereas veryintense wells may have their LEDs on during only 1% or less of theintegration time. On a time averaged basis, cross-talk between wellsbecomes negligible, and what little there is becomes highly consistentover time. Array pixels rarely become saturated. Since detection isalways at a large fraction of well capacity, array read noise becomesrelatively unimportant, and the system should become shot noise limitedfor all wells. Moreover, the resulting noise level will be the same aswhat would otherwise have been obtained for the baseline wells, and itis the noise at or near baseline which determines PCR quantitativeperformance. Some measurement time is saved by eliminating the multipleexposures, allowing either the PCR cycles to be speeded up or allowingfor more array reads to be summed for improved signal-to-noise ratios.

One enabler of this LED gating approach is that the PCR emission signalis inherently smooth as a function of cycle number. Thus, the change insignal from cycle to cycle is relatively small, except in the stronglyexponential phase. However, even in this phase the signal at the nextcycle can still be well predicted from the previous cycles. Thus aprediction algorithm can be used to decide what LED duty factor toemploy for each new measurement, to within say ±30%, with little or norisk of overexposure. Alternatively or additionally, a very shortpre-exposure could be used to determine the LED duty factor. Using thisscheme, the fluorescence signal used for PCR quantitation is composed oftwo multiplicative factors: the LED duty factor that was employed is themain component, with the array detector signal summed over the pixelsmaking up the region of interest (ROI) for a well acting as a vernieradjustment factor. The detector signal contributes almost all the noisesince LED timing can be highly accurate and reproducible.

Many detectors, as described herein capable of on-chip electronicshuttering, make use of microlenses to increase effective quantumefficiency. An important side effect of the use of micro lenses is toreduce the effective angular acceptance range of each pixel of thearray. In many examples, the microlenses are situated such that the peaksignal is obtained at normal incidence, and beyond a certain off-axisangle, symmetrically disposed about the normal, response falls offrapidly. Usually the angular width is different along the two orthogonalaxes of the sensor.

The effect of this microlensing shading is to place a fairly severelimit on the practical numerical aperture (NA) of the camera optics, andto require that for best results the lens be telecentric at the sensor.For a given sensor area (driven by economics), the area combined withthe limiting NA defines a practical maximum system etendue. Because ofthis, the multifunction mirror can be implemented in a system hereinwith minimal penalty.

That is, the etendue allowed by sensor microlensing is readilyaccommodated in a pupil shared system with up to at least 3 separateexcitation beams, without any compromise. Under these circumstances, useof dichroic beamsplitters (which incur numerous disadvantages) would notresult in any signal increase as compared to a multifunction mirror.

The excitation power which can be delivered to a well using theone-LED-per well as described herein is effectively the radiance of anLED die (limited by available LED technology at required wavelengths,more or less independent of die size), multiplied by the excitationoptics per-well etendue. This per-well etendue depends on the image sizeof the LED die in the well, as well as the pupil area. The actual sizeof the source LED die may not matter, although there are practicaloptically determined limits on how small the dies can be whilemaximizing the image size at each well.

When LEDs are widely spaced in the source array, using availablediscrete surface mount packages, the fractional area of the source arrayactually filled by the emitting dies is small. Using a simple objective,imaging the full LED array 1:1 onto the well plate, the die image sizesare quite small, a small fraction of the well diameter, and hence theexcitation power is less than it could be.

In some instances, the optics of the excitation optical path comprisecomposite optics, in which a simple objective images the full array,while a lenslet associated with each LED permits local magnification ofthe LED image so that a normally die size (typically 300 um square) canessentially fill the active portion of a microwell (˜2 mm diameter).With this solution, the NA at each source LED is relatively high, andthe system is designed to be telecentric at the dies. The beam from eachLED passes through the common excitation pupil; this can be accomplishedeither by use of a full-array diameter field lens or by displacingindividual lenslet centers with respect to die centers.

In some embodiments, a system herein comprises 2 arrays of LEDs, forexample, green and blue. In other embodiments, an array of LEDs caninclude LEDs of two different colors, for example, green and blue. Inyet other embodiments, any array of LEDs can include LEDs of more thantwo different colors. With distance, temperature can degrade LED signal,especially for green LEDs. Temperature reduction maximizes radiance formLED and can extend the time of which the well can be imaged with eachexcitation. Temperature control as provided herein also reduces signalvariance and improves measurements.

FIG. 11 illustrates a sample image from a 48-well sample plate,including well images 1102, as excited and detected using an opticalsystem as described herein. Each well is individually excited anddetected using the detector of the optical system.

FIG. 12 illustrates an example optical assembly as described hereincomprising a multifunction mirror 1202. In the example embodiment ofFIG. 12, both the excitation source assembly 1204 and the detectionassembly are in the same plane. The excitation source assembly, thedetection assembly, and the multifunction mirror are mounted on anoptical system baseplate 1208. Also demonstrated are optical filters1210 in the detection optical path that can be moved to change thefilter wavelength in the detection optical path.

FIG. 13 shows a top view of the optical system over the sample plate.For example in FIG. 13, the optical system comprises two LED arrays 1302as described herein that can simultaneously or sequentially excite thesamples in the thermal block by directing the energy through themultifunction mirror 1202. The emission from the sample plate on sampleblock 1306 is directed back through the multifunction mirror 1202 and tothe detector 1310 which is in the same plane and mounted on the sampleoptical plate as the LED arrays 1302 (e.g., the optical baseplate 1208of FIG. 12). Filters 1312 can be included in the excitation and/oremission paths, as illustrated in FIG. 13.

FIG. 14 demonstrates the detection optical path of the optical assembly.In the example embodiment, the detection optical path travels from themultifunction mirror through the detection optics 1402, through adetection optical filter 1404, and to the detector 1406. In anembodiment, the detector 1406 is a CCD camera.

FIG. 15 illustrates an excitation source assembly 1502 as describedherein. For example, the excitation source assembly 1502 can comprise anLED array. In an embodiment the LED array has the same number of LEDsources as there are wells in a sample plate. In another embodiment, theLED array has a different number of LEDs to the number of wells in asample plate. For example, two or more LEDs can correspond to anindividual sample plate. As another example, one LED can provide lightto two or more sample plates. In an embodiment, an LED array herein has48 LEDs. In an embodiment, a sample plate has 48 wells. As demonstratedin FIG. 15, the excitation source assembly can comprise a lenslet array1504 corresponding to the LED array.

FIG. 16 illustrates an example optical assembly as described hereincomprising a multifunction mirror 1602, excitation optics 1604, and anexcitation source assembly 1606. In an embodiment, the excitation sourceassembly 1606 comprises a lens array, an LED array, and backplate 1608on which the LED array is mounted. Using this configuration, the LEDarray can be cooled or temperature regulated by regulating thetemperature of the backplate 1608. As demonstrated in FIG. 16, both theexcitation optical path and the detection optical path are in the sameplane. In many embodiments, the optical path plane is parallel to thetop surface of the thermal block.

FIG. 17 illustrates an excitation source board 1702 on which an array ofLEDs can be mounted. Each LED is separately wired to the control boardand can be controlled individually by a computer system. In someinstances, each of the LEDs can be programmed to emit different amountsof energy. For example, if one well fluoresces more than another well,the excitation energy from the LED corresponding to that well can belowered to more easily compare the samples between each well.

In some instances, an excitation source is a flood light, for example, a100 watt halogen lamp. The light source can provide light at selectivewavelengths, coherent or incoherent. A mechanical or electronic shuttercan be used for blocking the source beam of the excitation source forobtaining dark data. The type of light source can also be a LED,projection lamp, or a laser, with appropriate optical elements.

In some instances, an excitation source can be used to provideexcitation beams to irradiate a sample solution containing one or moredyes. For example, two or more excitation beams having the same ordifferent wavelength emissions can be used such that each excitationbeam excites a different respective dye in the sample. The excitationbeam can be aimed from the light source directly at the sample, througha wall of a sample container containing the sample, or can be conveyedby various optical systems to the sample. An optical system can includeone or more of, for example, a mirror, a beam splitter, a fiber optic, alight guide, or combinations thereof.

According to various embodiments, excitation beams emitted from thelight source can diverge from the light source at an angle ofdivergence. The angle of divergence can be, for example, from about 5°to about 75° or more. The angle of divergence can be substantially wide,for example, greater than 45°, yet can be efficiently focused by use ofa lens, such as a focusing lens.

One or more filters, for example, a bandpass filter, can be used with alight source to control the wavelength of an excitation beam. One ormore filters can be used to control the wavelength of an emission beamemitted from an excited or other luminescent marker. One or moreexcitation filters can be associated with a light source to form theexcitation beam. One or more filters can be located between the one ormore light sources and a sample. One or more emission filters can beassociated with an emission beam from an excited dye. One or morefilters can be located between the sample and one or more emission beamdetectors. According to various embodiments, optics in both thedetection and excitation optical paths can include optical filters.Example filters can be conventional optical bandpass filters utilizingoptical interference films, each having a bandpass at a frequencyoptimum either for excitation of the fluorescent dye or its emission.Each filter can have very high attenuation for the other (non-bandpass)frequency, in order to prevent “ghost” images from reflected and straylight. For SYBR Green dye, for example, the excitation filter bandpasswavelength can center around 485 nm, and the emission filter bandpasswavelength can center around 555 nm. The beam splitter can transitionfrom reflection to transmission between these two, for example about 510nm, so that light less than this wavelength can be reflected and higherwavelength light can be passed through.

According to various embodiments, the excitation source can be a LightEmitting Diode (LED). The LED can be, for example, an Organic LightEmitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), ora Quantum dot based inorganic organic LED. The LED can include aphosphorescent OLED (PHOLED). As used herein, the terms excitationsource and light source are used interchangeably.

FIG. 18 illustrates an example LED 1802 as can be incorporated into asystem of the invention. The LED 1802 is mounted on an aluminumsubstrate 1804. The surface of the LED 1802 is coated with epoxy 1806 oranother polymer material to protect the diode. Because the aluminum isthermally conductive, the LED 1802 can be cooled or temperatureregulated through by cooling or regulating the aluminum substrate 1804.

In some instances, a light source can contain one Light Emitting Diode(LED) or an array of LEDs. According to various embodiments, each LEDcan be a high power LED that can emit greater than or equal to about 1mW of excitation energy. In various embodiments, a high power LED canemit at least about 5 mW of excitation energy. In various embodimentswherein the LED or array of LEDs can emit, for example, at least about50 mW of excitation energy, a cooling device such as, but not limitedto, a heat sink or fan can be used with the LED. An array ofhigh-powered LEDs can be used that draws, for example, about 10 watts ofenergy or less or about 10 watts of energy or more. The total power drawcan depend on the power of each LED and the number of LEDs in the array.The use of an LED array can result in a significant reduction in powerrequirement over other light sources, such as, for example, a 75 watthalogen light source or a 150 watt halogen light source. Example LEDarray sources are available. In some instances, LED light sources canuse about 1 microwatt of power or less, for example, about 1 mW, about 5mW, about 25 mW, about 50 mW, about 100 mW, about 500 mW about 1 W,about 5 W, about 50 W, or about 100 W or more, individually or when inused in an array. In some instances, the LED light sources use about 1microwatt to about 100 W of power.

In some embodiments, the optical assembly can be mounted over thethermal block containing the sample plate. In order to access the sampleplate when it is in the device, a movable lid (e.g., a hinged lid)comprising a heated lid for the sample plate can be moved without movingthe optical assembly or the thermal assembly to access the sample plate.In some instances, the movable lid is positioned to move around theoptical assembly. In addition, the movable lid can include a heated lidthat is configured to mate with the sample plate. The heated lid can beattached to the movable lid by springs or other materials such that whenthe movable lid is closed, the heated lid presses tightly against thethermal block to improve thermal contact. The heated lid can include anarray of holes therethrough aligned with the wells of the sample plate.Each hole can have a diameter about the same as the top diameter of awell.

Above each of the wells, multiple lenses or a single lens can bepositioned such that its focal point is directs energy into each of thewell. In some instances, the lens above the sample plate can be atelecentric optical system, a pixilated lens, or preferably a Fresnellens. In some embodiments, the lens above the wells is incorporated intothe movable lid.

FIG. 19 shows an example thermal cycling device with optical system asdescribed herein. The cover of the device covers the entire device andcan seal the thermal block area for protection while the assay is beingrun. As shown in FIG. 19, the optical assembly 1901 is mounted above thethermal block and is in a fixed position relative to the thermalassembly 1902. The heated lid 1904 is attached to the movable lid 1906,such that when the movable lid 1906 is open, the heated lid 1904 is notin contact with the sample plate 1908 and allows for placement andremoval of the sample plate 1908 from the device. When the movable lid1906 is closed, the heated lid 1904 is configured to couple with thesample plate 1908. The movable lid 1906 can move independently from therest of the system, such that it is the only moving part of the system(along with its components such as the heated lid 1904). In anotherembodiment, the movable lid 1906 (and its components) and a detectionfilter assembly are the only moving parts of the system.

FIG. 20 demonstrates an example thermal cycling device with opticalsystem as described herein with the movable lid open. The heated lid2002 is visible within the movable lid 2004 and is configured to couplewith the sample plate mounted on the thermal assembly 2006. The opticalassembly remains in a fixed position relative to the base 2008 of thedevice.

FIG. 21 illustrates an example device 2102 as described herein. Thedevice body 2104 comprises a movable lid 2106 comprising a grip 2108 andindicator lights 2110, a latch 2112, and cooling vents 2114. The movablelid 2106 opens to provide access to the thermal assembly and to allow asample holder to be placed within the device 2102. The movable lid 2106travels over the stationary optical system. In this manner, the opticalsystem is in a fixed position as compared to the thermal assembly anddoes not move. The movable lid 2106 can also comprise a heated lid thatcouple to the thermal block to prevent condensation on the sample holderduring thermal cycling. In some instances, the movable lid 2106comprises a lid compression plate that couples with the compressionplate on a thermal assembly to provide pressure to couple the heated lidto the sample holder. Cooling portals are also provided for the thermalassembly.

FIG. 22 illustrates an example device 2202 as described hereincomprising a device body 2204 and not displaying the cover to themovable lid. The movable lid plate 2206 is displayed in FIG. 22. Themovable lid plate 2206 comprises a holder 2208 for the heated lid thatcouples with a sample holder. Also displayed in FIG. 22 is a lid hinge2210 for the movable lid. In some instances, the lid hinge 2210 isspring loaded. In some instances, the lid hinge 2210 is motorized. Insome instances, the lid hinge 2210 is manually powered. The latch 2212of the body 2204 is used to secure and release the movable lid. Alsodemonstrated in FIG. 22 is a fan portal 2214 for mounting a cooling fanthat cools the excitation source assembly of the optical assemblythrough the cooling portal. Further illustrated are indicator lights2216.

FIG. 23 illustrates a movable lid plate 2302 that can be coupled with aheated lid 2304 that covers the optical assembly 2306. In manyembodiments, the optical assembly 2306 and the thermal assembly are bothstationary and the heated lid 2304, which comprises a cover for thesample plate, and a Fresnel lens 2308, can move over the opticalassembly and can function to couple the cover to the sample plate of thethermal assembly.

FIG. 24 demonstrates a thermal block 2402 and a compression plate 2404of a system as described herein. The compression plate 2404 has anaperture to provide access to the thermal block 2402 and is configuredto receive a cover or heated cover for the thermal block 2402. Thecompression plate 2404 can couple with the heated cover to provide closeor complete thermal contact.

FIG. 25 displays a system herein comprising an optical assembly 2502 anda movable lid plate 2504. The movable lid is not shown. In the exampleof FIG. 25, the optical assembly 2502 comprises a fan 2506 to providecooling for the excitation source. For example, if the excitation sourceis an LED array mounted on an aluminum backplate as described herein,the fan 2506 can cool the LED array.

Any combination of features of the optical systems described herein canbe implemented in connection with a thermal assembly. For example, athermal assembly can include a thermal block including the sample plate.Such an example system can also include: (a) two excitation sourceassemblies positioned in the excitation optical path, wherein anexcitation source assembly comprises one light emitting diode (LED)corresponding to each well on the sample plate; (b) a multifunctionmirror positioned in the excitation optical path to direct excitationenergy from each LED to the corresponding well of the sample plate andwherein the multifunction mirror is further positioned in the detectionoptical path to direct fluorescence emission from the sample plate to adetector; (c) two sets of excitation optics positioned in the excitationoptical path, a set of excitation optics comprising an excitation filterand two lenses, optionally, wherein the excitation filter is positionedbetween the two lenses; (d) a detector positioned in the detectionoptical path to detect the fluorescent emission that is directed fromthe multifunctional mirror; and (e) an emission filter slide positionedin the detection optical path between the multifunction mirror and thedetector, wherein the emission filter slide comprises at least oneemission filter. Moreover, in some instances, the thermal block of thisexample system can include a liquid composition. The liquid compositioncan be used to heat and cool samples to a substantially uniformtemperature, for example, as described in U.S. patent application Ser.No. 12/753,308, filed Apr. 2, 2010, the entire disclosure of which ishereby incorporated by reference.

In some cases, the system according to certain embodiments alsocomprises a thermal control unit or a thermal cycler to which the samplevessels in the multi-well plate can mate. The optical systems describedherein can be advantageous for measuring the state of reactions withinthe reaction vessels during and between reaction steps and cycleswithout having to remove the samples from the thermal cycling element.For example, the system can be used to measure polynucleotideamplification such as polymerase chain reaction (PCR) and real-timepolymerase chain reaction (RT-PCR) amplifications.

In embodiments described herein, the multiple temperature cyclescorrespond to multiple cycles of nucleic acid amplification. Nucleicacid amplification can comprise real-time PCR. For example, an apparatusor system according to certain embodiments can also be referred to as athermal cycler. Such a thermal cycler can include any combination offeatures described in U.S. patent application Ser. No. 12/753,308.

As described herein, a thermal block can include a liquid compositionthat can provide thermal contact between the heater and the sampleholder, and provide uniform heat transfer. As a result, the temperaturesof the samples within a sample holder can be substantially uniform. Thecombination of rapid temperature ramp rates and uniformity oftemperature decreases non-specific hybridization and significantlyincreases the specificity (for example, signal-to-noise ratio) ofamplification in PCR within individual wells as well as across multiplewells located in the same heat block (or reservoir). In anotherembodiment, the sample holder, alone or in combination with theapparatus, emits substantially all of a signal generated therein outthrough a discrete portion of the sample holder, for example, the top ofthe holder, whereby the emitted light can be collected by an opticalassembly. In yet another embodiment a light detector detectssubstantially all of the light emitted from a sample holder. In certainembodiments the reservoir is highly reflective and reflects lighttransmitted through the walls of a transparent sample holder back intothe sample holder. In this way, a greater proportion of a light signalgenerated inside the sample holder is emitted from a discrete portion ofthe sample holder, whereby it can be collected by the optical assembly.In an example, collecting light from a discrete location of the holdercan eliminate the necessity of removing the holder from the heat blockwhen performing real-time PCR. Accordingly, the apparatus herein isparticularly adapted for performing PCR (polymerase chain reaction),reverse transcription PCR and real-time PCR. In one embodiment anapparatus comprising a reservoir comprising a liquid composition ispowered by a battery or AC or DC current.

In addition to providing thermal cycling for PCR, an apparatus hereincan be used widely in the field of biotechnology and chemistry as isdiscussed herein. The use of a liquid composition as described canresult in a more uniform heat transfer and more rapid heating andcooling cycles than solid metal heat blocks, which in an example, canlead to lower error rates by DNA polymerases. Further, error rates maybe decreased during long amplifications, SNP identification andsequencing reactions, because of the enhanced thermal uniformity.

In some instances, the heater is a thermoelectric device. In otherinstances, the heater is a resistive device. An apparatus herein canalso comprise a cooler. In some instances, the heater and the cooler arethe same device, for example, a Peltier device.

A variety of heaters and coolers are known to a practitioner in the art.In one embodiment, a heater is a Peltier device or a resistive heater.In an embodiment, the thermal block is in thermal contact with aPeltier-effect thermoelectric device. In an alternative embodiment, theheater may be provided by extending a tube into the thermal blockthrough which hot or cold fluids can be pumped. In alternativeembodiments, the thermal block can be fitted with a heating and/orcooling coil, or with an electrical resistance heater arranged toprevent edge effects.

Peltier devices or elements, also known as thermoelectric (TE) modules,are small solid-state devices can function as heat pumps. A typicalPeltier unit is a few millimeters thick by a few millimeters to a fewcentimeters in a square or rectangular shape. It is a sandwich formed bytwo ceramic plates with an array of small Bismuth Telluride (Bi2Te3)cubes (“couples”) in between. When a DC current is applied heat is movedfrom one side of the device to the other where it can be removed by aheat sink. The “cold” side may be attached to a heat sink. If thecurrent is reversed the device changes the direction in which the heatis moved. Peltier devices lack moving parts, do not require refrigerant,do not produce noise or vibration, are small in size, have a long life,and are capable of precision temperature control. Temperature controlmay be provided by using a temperature sensor feedback (such as athermistor or a solid-state sensor) and a closed-loop control circuit,which may be based on a general purpose programmable computer.

In another embodiment the thermal cycler may further comprise anelectric resistance heater and a Peltier element used in combination toobtain the required speed of the temperature changes in the thermalblock and the required precision and homogeneity of the temperaturedistribution.

A heater as described herein may also comprise a heat sink as is knownto one skilled in the art. In one embodiment, a heat sink is a Peltierdevice, a refrigerator, an evaporative cooler, a heat pipe, a heat pump,or a phase change material. In one embodiment, the heat sink is athermoelectric device such as a Peltier device. The heat sink may alsobe a heat pipe, which is a sealed vacuum vessel with an inner wick whichserves to transfer heat by the evaporation and condensation of a fluid.Heat pipes which are suitable for use in the invention are disclosed,for example in WO 01/51209, U.S. Pat. No. 4,950,608, and U.S. Pat. No.4,387,762. Similarly suitable devices are produced by the companyThermacore (Lancester, USA) and sold under the trade name Therma-Base™.Additional devices for use as heat sinks are also described in U.S. Pat.No. 5,161,609 and U.S. Pat. No. 5,819,842.

In an alternative embodiment, a heater and sometimes the reservoir isdesigned to maintain different temperatures in different zones of thereservoir wells. This can allow different sample wells in differentzones to be cycled at different temperatures simultaneously. In oneembodiment the liquid metal or thermally conductive fluid heat block isa capable of maintaining a temperature gradient across 2, 3, 4, 5, 6 ormore zones. In one embodiment temperature gradients in excess of 0.1° C.to 20° C. across the reservoir can be achieved. In some embodiments theheat block will contain internal baffles or insulated walls which act toseparate different zones of the liquid composition from other zones.Each zone may further comprise an individual fluid stirrer. Further eachzone of the heat block may comprise individual heating and/or coolingelements such as a heat conduction element (wires, tubes), thin foiltype heater, Peltier elements or cooling units.

As described herein, the sample holder can be a multiwell plate. In someinstances, the multiwell plate has 16, 24, 48, 96, 384 or more samplewells. In some of these instances, an array of light sources, such asLEDs, has 16, 24, 48, 96, 384 or more corresponding light sources. Insome instances, the multiwell plate is a standard microwell plate forbiological analysis. For example, the multiwell plate can be plate usedfor PCR. In an embodiment, the multiwell plate has of 48 sample wells.The apparatus described herein can function to keep the temperature ofthe samples within each of the sample wells of a multiwell plate within±0.3° C. In other embodiments, the sample holder can be sample tubes,such as Eppendorf tubes.

As described herein, a sample holder can be reaction vessels of avariety of shapes and configurations. In an embodiment sample holder canbe used to contain reaction mixtures, such as PCR reaction mixtures,reverse transcription reaction mixtures, real-time PCR reactionmixtures, or any other reaction mixture which requires heating, coolingor a stable uniform temperature. In one embodiment the sample holder isround or tubular shaped vessels. In an alternative embodiment the sampleholder is oval vessels. In another embodiment the sample holder isrectangular or square shaped vessels. Any of the preceding embodimentsmay further employ a tapered, rounded or flat bottom. In yet anotherembodiment the sample holder is capillary tubes, such as clear glasscapillary tubes or coated capillary tubes, wherein the coating (forexample metal) increases internal reflectivity. In an additionalembodiment the sample holder is slides, such as glass slides. In anotherembodiment the sample holder is sealed at the bottom. In anotherembodiment the sample holder is coated, at least internally, with amaterial for preventing an amplicon from sticking to the sample holderwalls, such as a fluorinated polymer or BSA.

In one embodiment the sample holder is manufactured and used asindividual vessels. In another embodiment the sample holder is aplurality of vessels linked together in a horizontal series comprising amultiple of individual vessels, such as 2, 4, 6, 10, 12, 14 or 16 tubes.In yet another embodiment the sample holder is linked together to form asheet, plate or tray of vessels designed to fit into the top of theheating block of a thermal cycler so as to occupy some or all availablereaction wells. In one embodiment the holder is a microplate comprisingat least 6, wells, 12 wells, 24 wells, 36 wells, 48 wells, 54 wells, 60wells, 66 wells, 72 wells, 78 wells, 84 wells, 90 wells or 96 wells, 144wells, 192 wells, 384 wells, 768, or 1536 wells.

In one embodiment the sample holder has caps or a cover attached to theopen ends of sample wells or vessels. In one embodiment the sample wellsor vessels are designed to hold a maximum sample volume, such as 10 ul,20 ul, 30 ul, 40 ul, 50 ul, 60 ul, 70 ul, 80 ul, 90 ul, 100 ul, 200 ul,250 ul, 500 ul, 750 ul, 1000 ul, 1500 ul, 2000 ul, 5 mL, or 10 mL.

In some embodiments real-time polymerase chain reactions (PCR) areperformed in a sample holder manufactured from materials chosen fortheir optical clarity and for their known non-interaction with thereactants, such as glass or plastic. In one embodiment the sample holderis designed so that light can enter and leave through the top portion ofthe sample wells, which may be covered with a material at leastpartially transparent to light. In one embodiment the sample holder isdesigned so that light is directed to exit through a single surface,such as the top or bottom.

In other embodiments the sample holder is manufactured from materialsthat are substantially internally reflective, such as reflectiveplastic, coated plastic (such as with metal or other reflectivesubstances), coated glass (such as with metal or other reflectivesubstances), doped glass (manufactured with the addition of moleculesthat increase the reflectivity of the glass), or metal, including butnot limited to stainless steel, chromium, or other substantiallynon-reactive metals.

In an aspect, a method of heating a biological sample comprises:positioning a sample holder containing a biological sample into thermalcontact with an apparatus as described herein; and heating thebiological sample contained by the sample holder with the apparatus.

In an embodiment, the method comprises performing PCR on the biologicalsample. The heating can comprises thermally cycling the biologicalsample between about 50-65° C. and about 90 to 100° C. PCR processes andmethods are discussed in further detail herein.

In some instances, an apparatus herein maintains the temperature of aplurality of biological samples when heating. For example, a pluralityof biological samples can be heated to 95° C. from 60° C., and within 10s, each of the biological samples is maintained within ±0.3° C. of eachother. In an embodiment, a plurality of biological samples is maintainedwithin ±0.5° C., ±0.4° C., ±0.3° C., ±0.2° C., ±0.1° C., ±0.05° C., or±0.01° C. of each other. In an embodiment, a plurality of biologicalsamples are brought to a temperature within ±0.5° C., ±0.4° C., ±0.3°C., ±0.2° C., ±0.1° C., ±0.05° C., or ±0.01° C. within 30, 20, 10, 5, 3,2, 1, or 0.5 s after changing the temperature of the biological samplesby more than 5, 10, 20, 30, 40, or 50° C. When changing temperature ofbiological samples using, for example, thermal cycling, temperatureuniformity of a plurality of biological samples can be important forimproving the quality of any assay or reaction products.

As described, the sample holder can be a multiwell plate and the wellsof the multiwell plate contain the biological sample, wherein thebiological sample is a polynucleotide sample.

In some instances, a method herein comprises providing reagents forcarrying out PCR, and dyes for detecting the level of amplification tothe wells containing the biological sample, thereby creating a reactionmixture.

Heating can comprise cycling the temperature of reaction mixture in thewells to perform multiple amplification cycles. In some instances, eachof the amplification cycles comprise an annealing temperature and adenaturing temperature, and wherein the annealing (or denaturing orboth) temperature of each amplification cycle varies by less than ±0.3°C.

In some embodiments the uniformity of temperature of the liquidcomposition and reservoir is regulated by a step of a method herein ofcirculating the liquid composition in the reservoir. Circulation of theliquid metal or thermally conductive fluid can be created by naturalconvection or forced convection, such as by the intervention of a deviceincluding but not limited to a stir bar and a pump.

In some embodiments a method herein provides a thermal cycling ramp rateat a rate substantially faster than conventional metal heat blocks, suchas at a rate of at least 5-50.5° C. per second, including but notlimited to a range of at least 10-40° C. per second. In a relatedembodiment a method and apparatus herein can change temperature at arate substantially faster than conventional metal heat blocks whilemaintaining a more uniform temperature across the heat block and/orwithin a sample within said heat block. In one embodiment thetemperature of the biological samples in thermal contact with the heatblock can be measured with glass bead thermistors (Betatherm). Inanother embodiment an infrared camera is used to measure the temperatureof the samples. In another embodiment the temperature of the liquidsample is measured with an external probe.

In some instances, a method comprises thermally cycling a biologicalsample. In some instances, the thermal cycling of a biological samplecan occur faster than many current standard thermal cycling devices. Inan embodiment, an apparatus described herein comprising a reservoir anda stirring device can heat a PCR reaction from the annealing temperatureto the denaturing temperature of the reaction in less than 10, 5, 4, 3,2, 1, 0.5, 0.2, 0.1, or 0.05 s. In an embodiment, an apparatus describedherein comprising a reservoir and a stirring device can cool a PCRreaction from the denaturing temperature to the annealing temperature ofthe reaction in less than 10, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1, or 0.05 s.

A method herein can also further comprise optically measuring the dyesbetween or during each of a plurality of amplification cycles todetermine the level of amplification.

In an aspect, a method of heating a biological sample as disclosedherein comprises: positioning a sample holder into thermal contact witha heater, wherein the sample holder comprises at least about 16 wellscontaining a biological sample and is at least 1 cm in width; andheating the biological sample within the sample holder with the heater;wherein the temperature variance of the biological sample between eachof the at least about 16 wells is less than ±0.3° C. In some instances,the temperature variance is less than ±0.3° C. within 10 secondsimmediately after increasing or decreasing the temperature of thebiological sample more than 10° C. per second. In an embodiment, thesample holder is at least 0.1, 0.5, 1, 2, 3, 4, 5, or 10 cm in width. Inan embodiment, all the wells are at the same temperature at the sametime.

In various embodiments a control assembly is operatively linked to anapparatus or thermal cycler of the invention. Such a control assembly,for example, comprises a programmable computer comprising computerexecutable logic that functions to operate any aspect of the devices,methods and/or systems of the invention. For example, the controlassembly can turn on/off or actuate motors, fans, regulating circuits,stir bars, continuous flow devices and optical assemblies. The controlassembly can be programmed to automatically process samples, runmultiple PCR cycles, obtain measurements, digitize measurements intodata, convert data into charts/graphs and report.

Computers for controlling instrumentation, recording signals, processingand analyzing signals or data can be any of a personal computer (PC),digital computers, a microprocessor based computer, a portable computer,or other type of processing device. Generally, a computer comprises acentral processing unit, a storage or memory unit that can record andread information and programs using machine-readable storage media, acommunication terminal such as a wired communication device or awireless communication device, an output device such as a displayterminal, and an input device such as a keyboard. The display terminalcan be a touch screen display, in which case it can function as both adisplay device and an input device. Different and/or additional inputdevices can be present such as a pointing device, such as a mouse or ajoystick, and different or additional output devices can be present suchas an enunciator, for example a speaker, a second display, or a printer.The computer can run anyone of a variety of operating systems, such asfor example, anyone of several versions of Windows, or of Mac OS, or ofUnix, or of Linux.

In some embodiments, the control assembly executes the necessaryprograms to digitize the signals detected and measured from reactionvessels and process the data into a readable form (for example, table,chart, grid, graph or other output known in the art). Such a form can bedisplayed or recorded electronically or provided in a paper format.

In some embodiments, the control assembly controls regulating circuitslinked to the thermal elements so as to regulate/control cycles oftemperatures of an apparatus as described herein.

In further embodiments, for example in real-time PCR, the controlassembly generates the sampling strobes of the optical assembly, therate of which is programmed to run automatically. The timing can beadjustable for shining a light sources and operating a detector todetect and measure signals (for example, fluorescence).

In another embodiment an apparatus comprising a control assembly furthercomprises a means for moving sample vessels into apertures, such aswells in the receptacle of a heat block comprising a liquid composition.In an embodiment said means could be a robotic system comprising motors,pulleys, clamps and other structures necessary for moving samplevessels.

In some aspects of the invention, the devices/systems of the inventionare operatively linked to a robotics sample preparation and/or sampleprocessing unit. For example, a control assembly can provide a programto operate automated collection of samples, adding of reagents tocollection tubes, processing/extracting nucleic acids from said tubes,optionally transferring samples to new tubes, adding necessary reagentsfor a subsequent reaction (for example, PCR or sequencing), andtransferring samples to a thermal cycler according to the invention.

A system as configured herein can be used for disease diagnosis, drugscreening, genotyping individuals, phylogenetic classification,environmental surveillance, parental and forensic identification amongstother uses. Further, nucleic acids can be obtained from any source forexperimentation. For example, a test sample can be biological and/orenvironmental samples. Biological samples may be derived from human,other animals, or plants, body fluid, solid tissue samples, tissuecultures or cells derived therefrom and the progeny thereof, sections orsmears prepared from any of these sources, or any other samplessuspected to contain the target nucleic acids. Example biologicalsamples are body fluids including but not limited to blood, urine,spinal fluid, cerebrospinal fluid, sinovial fluid, ammoniac fluid,semen, and saliva. Other types of biological sample may include foodproducts and ingredients such as vegetables, dairy items, meat, meatby-products, and waste. Environmental samples are derived fromenvironmental material including but not limited to soil, water, sewage,cosmetic, agricultural, industrial samples, air filter samples, and airconditioning samples.

An apparatus herein can be used in any protocol or experiment thatrequires either thermal cycling or a heat block that can accuratelymaintain a uniform temperature. For example said thermal cycler can beused for polymerase chain reaction (PCR), quantitative polymerase chainreaction (qPCR), nucleic acid sequencing, ligase chain polymerase chainreaction (LCR-PCR), reverse transcription PCR reaction (RT-PCR), singlebase extension reaction (SBE), multiplex single base extension reaction(MSBE), reverse transcription, and nucleic acid ligation.

PCR reaction conditions typically comprise either two or three stepcycles. Two step cycles have a denaturation step followed by ahybridization/elongation step. Three step cycles comprise a denaturationstep followed by a hybridization step during which the primer hybridizesto the strands of DNA, followed by a separate elongation step. Thepolymerase reactions are incubated under conditions in which the primershybridize to the target sequences and are extended by a polymerase. Theamplification reaction cycle conditions are selected so that the primershybridize specifically to the target sequence and are extended.

Successful PCR amplification requires high yield, high selectivity, anda controlled reaction rate at each step. Yield, selectivity, andreaction rate generally depend on the temperature, and optimaltemperatures depend on the composition and length of the polynucleotide,enzymes and other components in the reaction system. In addition,different temperatures may be optimal for different steps. Optimalreaction conditions may vary, depending on the target sequence and thecomposition of the primer. Thermal cyclers may be programmed byselecting temperatures to be maintained, time durations for each cycle,number of cycles, rate of temperature change and the like.

Primers for amplification reactions can be designed according to knownalgorithms. For example, algorithms implemented in commerciallyavailable or custom software can be used to design primers foramplifying desired target sequences. Typically, primers can range fromleast 12 bases, more often 15, 18, or 20 bases in length but can rangeup to 50+ bases in length. Primers are typically designed so that all ofthe primers participating in a particular reaction have meltingtemperatures that are within at least 5° C., and more typically within2° C. of each other. Primers are further designed to avoid priming onthemselves or each other. Primer concentration should be sufficient tobind to the amount of target sequences that are amplified so as toprovide an accurate assessment of the quantity of amplified sequence.Those of skill in the art will recognize that the amount ofconcentration of primer will vary according to the binding affinity ofthe primers as well as the quantity of sequence to be bound. Typicalprimer concentrations will range from 0.01 uM to 0.5 uM.

In one embodiment, an apparatus herein may be used for PCR, either aspart of a thermal cycler or as a heat block used to maintain a singletemperature. In a typical PCR cycle, a sample comprising a DNApolynucleotide and a PCR reaction cocktail is denatured by treatment ina thermal block at about 90-98° C. for 10-90 seconds. The denaturedpolynucleotide is then hybridized to oligonucleotide primers bytreatment in a thermal block of the invention at a temperature of about30-65° C. for 1-2 minutes. Chain extension then occurs by the action ofa DNA polymerase on the polynucleotide annealed to the oligonucleotideprimer. This reaction occurs at a temperature of about 70-75° C. for 30seconds to 5 minutes in the thermal block. Any desired number of PCRcycles may be carried out depending on variables including but notlimited to the amount of the initial DNA polynucleotide, the length ofthe desired product and primer stringency.

In another embodiment, the PCR cycle comprises denaturation of the DNApolynucleotide at a temperature of 95° C. for about 1 minute. Thehybridization of the oligonucleotide to the denatured polynucleotideoccurs at a temperature of about 37-65° C. for about one minute. Thepolymerase reaction is carried out for about one minute at about72.degree. C. All reactions are carried out in a multiwell plate whichis inserted into the wells of a receptacle in a thermal block of theinvention. About 30 PCR cycles are performed. The above temperatureranges and the other numbers are not intended to limit the scope of theinvention. These ranges are dependent on other factors such as the typeof enzyme, the type of container or plate, the type of biologicalsample, the size of samples, etc. One of ordinary skill in the art willrecognize that the temperatures, time durations and cycle number canreadily be modified as necessary.

Reverse transcription refers to the process by which mRNA is copied tocDNA by a reverse transcriptase (such as Moloney murine leukemia virus(MMLV) transcriptase avian myeloblastosis virus (AMV) transcriptase or avariant thereof) composed using an oligo dT primer or random oligomers(such as a random hexamer or octamer). In real-time PCR, a reversetranscriptase that has an endo H activity is typically used. Thisremoves the mRNA allowing the second strand of DNA to be formed. Reversetranscription typically occurs as a single step before PCR. In oneembodiment the RT reaction is performed in a thermal block of theinvention by incubating an RNA sample a transcriptase the necessarybuffers and components for about an hour at about 37° C., followed byincubation for about 15 minutes at about 45° C. followed by incubationat about 95° C. The cDNA product is then removed and used as a templatefor PCR. In an alternative embodiment the RT step is followedsequentially by the PCR step, for example in a one-step PCR protocol. Inthis embodiment all of the reaction components are present in the samplevessel for the RT step and the PCR step. However, the DNA polymerase isblocked from activity until it is activated by an extended incubation at95° C. for 5-10 minutes. In one embodiment the DNA polymerase is blockedfrom activity by the presence of a blocking antibody that is permanentlyinactivated during the 95° C. incubation step.

In molecular biology, real-time polymerase chain reaction, also calledquantitative real time polymerase chain reaction (QRT-PCR) or kineticpolymerase chain reaction, is used to simultaneously quantify andamplify a specific part of a given DNA molecule. It is used to determinewhether or not a specific sequence is present in the sample; and if itis present, the number of copies in the sample. It is the real-timeversion of quantitative polymerase chain reaction (Q-PCR), itself amodification of polymerase chain reaction.

The procedure follows the general pattern of polymerase chain reaction,but the DNA is quantified after each round of amplification; this is the“real-time” aspect of it. In one embodiment the DNA is quantified by theuse of fluorescent dyes that intercalate with double-strand DNA. In analternative embodiment modified DNA oligonucleotide probes thatfluoresce when hybridized with a complementary DNA are used to quantifythe DNA.

In another embodiment real-time polymerase chain reaction is combinedwith reverse transcription polymerase chain reaction to quantify lowabundance messenger RNA (mRNA), enabling a researcher to quantifyrelative gene expression at a particular time, or in a particular cellor tissue type.

In certain embodiments, the amplified products are directly visualizedwith detectable label such as a fluorescent DNA-binding dye. In oneembodiment the amplified products are quantified using an intercalatingdye, including but not limited to SYBR green, SYBR blue, DAPI, propidiumiodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine,acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin. For example, a DNA binding dye such as SYBRGreen binds all double stranded dsDNA and an increase in fluorescenceintensity is measured, thus allowing initial concentrations to bedetermined. A standard PCR reaction cocktail is prepared as usual, withthe addition of fluorescent dsDNA dye and added to a sample. Thereaction is then run in a liquid heatblock thermal cycler, and aftereach cycle, the levels of fluorescence are measured with a camera. Thedye fluoresces much more strongly when bound to the dsDNA (i.e. PCRproduct). Because the amount of the dye intercalated into thedouble-stranded DNA molecules is typically proportional to the amount ofthe amplified DNA products, one can conveniently determine the amount ofthe amplified products by quantifying the fluorescence of theintercalated dye using the optical systems of the present invention orother suitable instrument in the art. When referenced to a standarddilution, the dsDNA concentration in the PCR can be determined. In someembodiments the results obtained for a sequence of interest may benormalized against a stably expressed gene (“housekeeping gene”) such asactin, GAPDH, or 18s rRNA.

In an embodiment, any of the methods and systems herein can be used forhigh resolution melt (HRM) analysis of a sample or set of samples. Inother instances, a system can include multiple reference genes to pursueexpert analysis.

In various embodiments, labels/dyes are detected by systems or devicesof the invention. The term “label” or “dye” refers to any substancewhich is capable of producing a signal that is detectable by visual orinstrumental means. Various labels suitable for use in the presentinvention include labels which produce signals through either chemicalor physical means, such as fluorescent dyes, chromophores,electrochemical moieties, enzymes, radioactive moieties, phosphorescentgroups, fluorescent moieties, chemiluminescent moieties, or quantumdots, or more particularly, radiolabels, fluorophore-labels, quantumdot-labels, chromophore-labels, enzyme-labels, affinity ligand-labels,electromagnetic spin labels, heavy atom labels, probes labeled withnanoparticle light scattering labels or other nanoparticles, fluoresceinisothiocyanate (FITC), TRITC, rhodamine, tetramethylrhodamine,R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin(APC), probes such as Taqman probes, TaqMan Tamara probes, TaqMan MGBprobes or Lion probes (Biotools), fluorescent dyes such as SYBR Green I,SYBR Green II, SYBR gold, Cell Tracker Green, 7-AAD, ethidium homodimerI, ethidium homodimer II, ethidium homodimer III or ethidium bromide,epitope tags such as the FLAG or HA epitope, and enzyme tags such asalkaline phosphatase, horseradish peroxidase, I²-galactosidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase and haptenconjugates such as digoxigenin or dinitrophenyl, or members of a bindingpair that are capable of forming complexes such as streptavidin/biotin,avidin/biotin or an antigen/antibody complex including, for example,rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, erythrosin, coumarin,methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow,Cascade Blue, dichlorotriazinylamine fluorescein, dansyl chloride,phycoerythrin, fluorescent lanthanide complexes such as those includingEuropium and Terbium, Cy3, Cy5, molecular beacons and fluorescentderivatives thereof, a luminescent material such as luminol; lightscattering or plasmon resonant materials such as gold or silverparticles or quantum dots; or radioactive material including 14C, 123I,124I, 125I, 131I, Tc99m, 35S or 3H; or spherical shells, and probeslabeled with any other signal generating label known to those of skillin the art. For example, detectable molecules include but are notlimited to fluorophores as well as others known in the art as described,for example, in Principles of Fluorescence Spectroscopy, Joseph R.Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6thEdition of the Molecular Probes Handbook by Richard P. Hoagland.

Intercalating dyes are detected using the devices of the inventioninclude but are note limited to phenanthridines and acridines (forexample, ethidium bromide, propidium iodide, hexidium iodide,dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, andACMA); some mirror grove binders such as indoles and imidazoles (forexample, Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); andmiscellaneous nucleic acid stains such as acridine orange (also capableof intercalating), 7-AAD, actinomycin D, LDS751, andhydroxystilbamidine. All of the aforementioned nucleic acid stains arecommercially available from suppliers such as Molecular Probes, Inc.

Still other examples of nucleic acid stains include the following dyesfrom Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24,-21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).Other detectable markers include chemiluminescent and chromogenicmolecules, optical or electron density markers, etc.

As noted above in certain embodiments, labels comprise semiconductornanocrystals such as quantum dots (i.e., Qdots), described in U.S. Pat.No. 6,207,392. Qdots are commercially available from Quantum DotCorporation. The semiconductor nanocrystals useful in the practice ofthe invention include nanocrystals of Group II-VI semiconductors such asMgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixedcompositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof. The use of Group IV semiconductors such asgermanium or silicon, or the use of organic semiconductors, may also befeasible under certain conditions. The semiconductor nanocrystals mayalso include alloys comprising two or more semiconductors selected fromthe group consisting of the above Group III-V compounds, Group II-VIcompounds, Group IV elements, and combinations of same.

In addition to various kinds of fluorescent DNA-binding dye, otherluminescent labels such as sequence specific probes can be employed inthe amplification reaction to facilitate the detection andquantification of the amplified product. Probe based quantitativeamplification relies on the sequence-specific detection of a desiredamplified product. Unlike the dye-based quantitative methods, itutilizes a luminescent, target-specific probe (for example, TaqMan®probes) resulting in increased specificity and sensitivity. Methods forperforming probe-based quantitative amplification are well establishedin the art and are taught in U.S. Pat. No. 5,210,015.

In another embodiment fluorescent oligonucleotide probes are used toquantify the DNA. Fluorescent oligonucleotides (primers or probes)containing base-linked or terminally-linked fluors and quenchers arewell-known in the art. They can be obtained, for example, from LifeTechnologies (Gaithersburg, Md.), Sigma-Genosys (The Woodlands, Tex.),Genset Corp. (La Jolla, Calif.), or Synthetic Genetics (San Diego,Calif.). Base-linked fluors are incorporated into the oligonucleotidesby post-synthesis modification of oligonucleotides that are synthesizedwith reactive groups linked to bases. One of skill in the art willrecognize that a large number of different fluorophores are available,including from commercial sources such as Molecular Probes, Eugene,Oreg. and other fluorophores are known to those of skill in the art.Useful fluorophores include: fluorescein, fluorescein isothiocyanate(FITC), carboxy tetrachloro fluorescein (TET), NHS-fluorescein, 5 and/or6-carboxy fluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein,5-{[2(and 3)-5-(Acetylmercapto)-succinyl]amino}fluorescein(SAMSA-fluorescein), and other fluorescein derivatives, rhodamine,Lissamine rhodamine B sulfonyl chloride, Texas red sulfonyl chloride, 5and/or 6 carboxy rhodamine (ROX) and other rhodamine derivatives,coumarin, 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-aceticacid (AMCA), and other coumarin derivatives, BODIPY™ fluorophores,Cascade Blue™ fluorophores such as 8-methoxypyrene-1,3,6-trisulfonicacid trisodium salt, Lucifer yellow fluorophores such as3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins derivatives,Alexa fluor dyes (available from Molecular Probes, Eugene, Oreg.) andother fluorophores known to those of skill in the art. For a generallisting of useful fluorophores, see also Hermanson, G. T., BIOCONJUGATETECHNIQUES (Academic Press, San Diego, 1996).

Embodiments using fluorescent reporter probes produce accurate andreliable results. Sequence specific RNA or DNA based probes are used tospecifically quantify the probe sequence and not all double strandedDNA. This also allows for multiplexing—assaying for several genes in thesame reaction by using specific probes with different colored labels.

In one embodiment PCR is carried out in a device of the inventionconfigured as a thermal cycler. In an embodiment, the thermal cyclerfurther comprises an optical assembly. In another embodiment the thermalblock of the thermal cycler rapidly and uniformly modulates thetemperature of samples contained within sample vessels to allowdetection of amplification products in real time. In another embodimentthe detection is via a non-specific nucleic acid label such as anintercalating dye, wherein the signal index, or the positivefluorescence intensity signal generated by a specific amplificationproduct is at least 3 times the fluorescence intensity generated by aPCR control sample, such as about 3.5, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, or 11. In an embodiment the thermal cycler maymodulate the sample temperature by more than 10° C. per second, such as10.5° C. per second.

In one embodiment an RNA based probe with a fluorescent reporter and aquencher held in adjacent positions is used. The close proximity of thereporter to the quencher prevents its fluorescence; it is only after thebreakdown of the probe that the fluorescence is detected. This processdepends on the 5′ to 3′ exonuclease activity of the polymerase used inthe PCR reaction cocktail.

Typically, the reaction is prepared as usual, with the addition of thesequence specific labeled probe the reaction commences. Afterdenaturation of the DNA the labeled probe is able to bind to itscomplementary sequence in the region of interest of the template DNA.When the PCR reaction is heated to the proper extension temperature bythe thermal block, the polymerase is activated and DNA extensionproceeds. As the polymerization continues it reaches the labeled probebound to the complementary sequence of DNA. The polymerase breaks theRNA probe into separate nucleotides, and separates the fluorescentreporter from the quencher. This results in an increase in fluorescenceas detected by the optical assembly. As PCR progresses more and more ofthe fluorescent reporter is liberated from its quencher, resulting in awell defined geometric increase in fluorescence. This allows accuratedetermination of the final, and initial, quantities of DNA.

In various applications, devices of the invention can be utilized for invitro diagnostic uses, such as detecting infectious or pathogenicagents. In one embodiment, PCR is conducted using a device of theinvention to detect various such agents, which can be any pathogenincluding without any limitation bacteria, yeast, fungi, virus,eukaryotic parasites, etc; infectious agent including influenza virus,parainfluenza virus, adenovirus, rhinovirus, coronavirus, hepatitisviruses A, B, C, D, E, etc, HIV, enterovirus, papillomavirus,coxsackievirus, herpes simplex virus, or Epstein-Barr virus; bacteriaincluding Mycobacterium, Streptococcus, Salmonella, Shigella,Staphylcococcus, Neisseria, Pseudomonads, Clostridium, or E. coli. Itwill be apparent to one of skill in the art that the PCR, sequencingreactions and related processes are readily adapted to the devices ofthe invention for use to detect any infectious agents.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A system comprising: a thermal assemblyconfigured to hold a sample plate; an optical assembly in a fixedposition relative to the thermal assembly, the optical assemblycomprising: at least one array of excitation sources configured to emitexcitation energy along an excitation optical path; and a detectorconfigured to receive emission energy along a detection optical path,wherein the excitation optical path and detection optical path are inthe same plane.
 2. The system of claim 1, wherein the system furthercomprises one or more of a multifunction mirror; an excitation sourceassembly comprising an array of excitation sources; an emission filterslide; a detection lens assembly; a set of excitation optics; or acontrol assembly.
 3. The system of claim 2, wherein the system comprisesa multifunction mirror, two excitation source assemblies, two sets ofexcitation optics and a detector.
 4. The system of claim 3, wherein, inthe excitation optical path, an excitation source assembly and a set ofexcitation optics is positioned on one side of the multifunction mirrorand another excitation source assembly and another set of excitationoptics is positioned on the other side of the multifunction mirror. 5.The system of claim 2, wherein the excitation source assembly ispositioned in the excitation optical path and wherein the excitationsource assembly comprises individual light emitting diodes (LEDs)corresponding to individual wells on the sample plate.
 6. The system ofclaim 5, wherein the excitation source assembly further comprises an LEDarray comprising the LEDs.
 7. The system of claim 6, wherein theexcitation source assembly further comprises a lenslet array comprisingindividual lenslets corresponding to individual LEDs in the LED array.8. The system of claim 2, wherein the multifunction mirror is positionedin the excitation optical path to direct excitation energy fromindividual LEDs to the corresponding wells of the sample plate and isfurther positioned in the detection optical path to direct emissionenergy from the sample plate to a detector.
 9. The system of claim 2,wherein the detector is positioned in the detection optical path and iscapable of detecting emission energy from the sample plate.
 10. Thesystem of claim 9, wherein the emission energy is fluorescent emissionand the fluorescent emission is directed to the detector from themultifunction mirror.
 11. The system of claim 2, wherein the systemfurther comprises an emission filter slide comprising at least oneemission filter.
 12. The system of claim 11, wherein the emission filterslide is positioned in the detection optical path between themultifunction mirror and the detector.
 13. The system of claim 12,wherein the emission filter slide comprises four emission filters, eachemission filter filtering a different wavelength.
 14. The system ofclaim 13, wherein the system further comprises an emission filter motorfor moving the emission filter slide.
 15. The system of claim 2, whereinthe set of excitation optics comprises an excitation filter and twolenses.
 16. The system of claim 1, wherein the system further comprisesa movable lid that is positioned to move around the optical assembly andwherein the movable lid comprises a heated lid configured to mate withthe sample plate.
 17. The system of claim 1, wherein the thermalassembly comprises a thermal block comprising the sample plate.
 18. Thesystem of claim 17, wherein a liquid composition occurs within thethermal block and external to the sample plate.
 19. The system of claim1, further comprising (a) at least one excitation source assemblypositioned in the excitation optical path, wherein the excitation sourceassembly comprises individual light emitting diodes (LEDs) correspondingto individual wells on the sample plate; (b) a multifunction mirrorpositioned in the excitation optical path to direct excitation energyfrom individual LEDs to the corresponding wells of the sample plate andwherein the multifunction mirror is further positioned in the detectionoptical path to direct fluorescence emission from the sample plate tothe detector; and (c) an emission filter slide positioned in thedetection optical path between the multifunction mirror and thedetector, wherein the emission filter slide comprises at least oneemission filter.
 20. The system of claim 19, wherein the excitationoptical path comprises two excitation source assemblies and two sets ofexcitation optics.
 21. The system of any one of claim 20, wherein eachset of excitation optics comprises an excitation filter and two lenses.22. The system of claim 21, wherein each excitation source assemblycomprises an LED backplate to which an LED array comprising the LEDs ismounted, a lenslet array comprising individual lenslets corresponding toindividual LEDs in the LED array, wherein the lenslet array ispositioned to transmit the excitation energy from the LEDs on the LEDarray to the multifunction mirror and an excitation source coolingportal positioned to cool the LED backplate.
 23. The system of claim 19,wherein the emission filter slide comprises four emission filters, eachemission filter filtering a different wavelength.
 24. The system ofclaim 23, wherein the system further comprising an emission filter motorfor moving the emission filter slide.
 25. The system of claim 19,further comprising at least one detection lens assembly positionedbetween the emission filter slide and the detector.
 26. The system ofclaim 19, wherein the system further comprises a movable lid that ispositioned to move around the optical assembly and wherein the movablelid comprises a heated lid configured to mate with the sample plate. 27.The system of claim 19, wherein the thermal assembly comprises a thermalblock comprising the sample plate.
 28. The system of claim 27, wherein aliquid composition occurs within the thermal block and external to thesample plate.